ZNF536 dysfunction enhances spontaneous differentiation of the SH-SY5Y cell line into a neuronal-like phenotype

TL;DR

This study shows that dysfunction of the ZNF536 gene causes SH-SY5Y cells to differentiate into neuron-like cells, suggesting a possible role in schizophrenia.

Contribution

A novel cellular model was developed to study ZNF536's role in schizophrenia pathogenesis using CRISPR/Cas9.

Findings

ZNF536 interacts with schizophrenia risk loci, indicating its involvement in the disorder.

ZNF536 deletion in SH-SY5Y cells leads to slowed growth and spontaneous neuronal differentiation.

CRISPR/Cas9 successfully generated ZNF536 heterozygous clones for further study.

Abstract

Schizophrenia (SZ) is a common psychiatric neurodevelopmental disorder with a complex genetic architecture. Genomic association studies indicate the involvement of transcription factors in the pathogenesis of SZ. A recent GWAS showed a significant association of ZNF536 with SZ. To date, the molecular functions of ZNF536 are poorly understood and its possible role in the pathogenesis of SZ is unclear.

The aim of this work was to develop a model cell line for study ZNF536-mediated pathogenic mechanisms associated with SZ.

To assess the spatial interaction of ZNF536 with SZ risk loci, we used the Capture-C method. For ZNF536 deletion, SH-SY5Y was sequentially transduced with two lentiviral vectors. The first expressed Cas9 under the control of a tetracycline regulated promoter and the second expressed a pair of sgRNAs for ZNF536 deletion. Puromycin was used to select transduced cells. Stably transduced cells were then treated with oxytetracycline to induce Cas9 expression. In parallel, SH-SY5Y were transduced with lentiviral constructs of Cas9 and sgRNA carrying a spacer lacking targets in the human genome to obtain a negative control. Individual clones were obtained by the limiting dilution method. The ZNF536 deletion was confirmed by PCR and Sanger sequencing.

A spatial interaction of ZNF536 with SZ risk loci was found, suggesting its involvement in SZ pathogenesis. Using the CRISPR/Cas9 system, we obtained several clones with heterozygous deletion of ZNF536. We observed that their growth and proliferation were significantly slowed down. In addition, the mutant clones spontaneously differentiate into a neuron-like phenotype in low-serum medium.

We established a cellular model to study ZNF536-mediated mechanisms associated with SZ.

None Declared